BioPack is a collection of pulse sequences, macros, and parameter sets distributed by Varian as an add-on package to VNMRJ. The idea behind it is the implementation of reproducible and predictable biomolecular NMR experiments that are easy to setup and calibrate. BioPack is installed in all the spectrometers at the Biomolecular NMR facility and gets updated often due to the frequent addition of new pulse sequences. You can access any of these experiments from the
in each user account. This Probe File is a database of calibrated pulse widths, powers, and other parameters used by many of the BioPack experiments. When an experiment is called, a standard parameter set is loaded and then several macros update them with the values saved within the Probe File. Each user account has a
(cold probe) probe file that I generated with the autocalibration routine using a 0.5mM sample of
N-labeled Gb1 in 50mM NaPi, 50mM NaCl, pH = 6.5. Many of the calibrated parameters in these files are not sample dependent, however, the hard 90° pulses (specially for proton) will be different from protein to protein and have to be calibrated before running any experiment. Each user can create his/her own probe file for his/her favorite protein by copying and renaming the HCN or HCN_cold probe file (these files live under the
directory) and updating the calibrations either manually or with the autocalibration routines in the
tab.
If you want to learn more about what’s behind the scenes in BioPack please read the on-line documentation found under the VNMRJ
Recording a 1H/15N-HSQC experiment
Finding tof manually
-Type
water in the command line to load a 1D water suppression proton NMR experiment.
-Type
nt=1 gain=0
-Array
tof and
satfrq together around the value shown for
tof in the standard parameter set. For example, if
tof=-170 type
array(‘tof’,20,-160,-1) satfrq=tof array=’(tof,satfrq)’ go
-Type
wft(1) av vsadj to process the first spectrum, display it in absolute value mode, and adjust its vertical scale to fit in the graphics canvas.
-Type
wft dssh dssl to display the array after the experiment finishes.
-Select the
Text Output folder under the
Acquire tab and type
da to display the array. Record the tof value for the spectrum with the smaller water signal.
Finding tof automatically
-Type
BPautopresat in the command line. This macro will determine
tof the same way as described above and it will run a 1D proton spectrum with the optimized value afterwards. You can find this value by typing
tof? in the command line or by selecting the
Text Output folder under the
Acquire tab and typing
dg in the command line.
Calibrating the hard pulses
The following lines describe how to calibrate the proton and nitrogen hard pulses using the gNhsqc experiment. Please look through the experiment manual for further details and learn what are some of the parameters doing. You can access the gNhsqc manual by typing
man(‘gNhsqc’) in the command line and selecting the
Text Out folder under the
Acquire tab.
The proton 90° pulse:
pw/tpwr
-Type
gNhsqc in the command line to load the
1H,
15N-HSQC parameters.
-Input the optimized value of
tof by typing
tof=optimized value.
-If your sample’s concentration is > 250mM and the molecular weight is < 15-20 KDa, nt=4 or 8 should be enough to quickly see the amide signals. Increase this number to a larger multiple of 4 for lower protein concentrations.
-Type
calH=1,1.8,2,2.2 and then
go to start the experiment. If you see an ADC Overflow error reduce the gain value until the error no longer appears.
-Type
wft to process the first spectrum, manually phase it positive or type
aph0, and adjust the vertical scale with
vsadj.
-Type
vp=50 dssh to display the array in the middle of the graphics canvas.
If the third spectrum has no intensity, the proton pulsewidth (
pw) is exactly 90°. However, if there is positive intensity, then the proton pulsewidth is too short and needs to be increased. On the other hand, if there is negative intensity then the pulsewidth is too long and has to be reduced. Adjust the
pw value and type
go again to re-run the experiment. Alternatively, you can type
calH=2 and array
pw to find the null as shown below.
Acceptable target values for
pw are 6ms-9ms for room temperature probes. Varian recommends the use of
pw > 8ms for
Cold Probes. On occasions, depending on the amount of salt in your sample, the optimum pulsewidth falls outside these ranges. If that’s the case, you have to increase or decrease the value of
tpwr.
Warning: Observe the maximum transmitter power (
tpwr) values for Cold Probes. These are posted on the computer monitors. Too much power can damage the probe!!!.
The nitrogen 90° pulse:
pwN/pwNlvl
-After calibrating the proton 90° pulse set
pw and
tpwr to their optimum values and
calH=1.
-Type
calN=1,1.8,2,2.2 and
go.
-Type
wft dssh to display the arrayed spectra. Again, the best nitrogen 90° pulse will be when the third spectrum (
calN=2) has the lowest intensity. However, in contrast to the proton
pw calibration, a complete null will be difficult to reach. Instead, you can look for the
pwN value that shows similar signal intensities, with opposite signs, in the spectra of
calN=1.8 and
calN=2.2. Alternatively, you can set
calN=2, array
pwN, and look for the spectrum with minimum signal intensity.
-Set
calN=1 and
pwN to its optimized value.
.
Running the
1H,
15N-HSQC experiment after calibrating
tof and hard pulses.
-Begin by running a 1D version of the experiment to make sure you get a spectrum and no parameter has been set incorrectly. A typical spectrum of a well folded, globular protein looks like the figure below.
-Adjust
sw1 for the correct spectral width in the indirectly detected dimension.
-Set
ni=64 or bigger (for better resolution) in the nitrogen dimension.
-Type
phase=1,2, ss=32 if using a RT probe, or
ss=256 if using a Cold Probe.
-Type
time to find out how long the experiment will last.
-Review all your parameters and if everything looks right type
go to start it.
-You can type
psnaf2 after four FIDs have been collected to obtain a nice printout of the experimental parameters for your lab notebook.
-Save the experiment after it finishes by typing
svf(‘data/filename’).
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Take a tour
SOLOMON ISLANDS
HONIARA
Malaita, Solomon Islands ...
.
.
Gizo,
on Ghizo Island, is the capital of the Solomon Islands’ far-flung
Western Province, a paradise of coral cays, atolls, lagoons and volcanic
islands east of Papua New Guinea where, on a rainy day in late July,
crowds flocked to the local netball court for the opening of the
inaugural Akuila Talasasa Arts Festival.
Motorised canoes lined up in Gizo Harbour near the daily marketplace.
.
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You mi